Methods and compositions for improving the nutritive value of foods

ABSTRACT

Mutant microorganisms comprising Lactobacillus fermentum Lex +  which are obtained from Lactobacillus fermentum produce lysine in a significantly greater quantity than the wildtype microorganism. The microorganism is added to a sourdough starter to produce bread of increased nutritive content, such as flat bread. Freeze-dried cultures of the microorganism may be added to cereal grains such as wheat in bulk to increase the basic nutritive protein quality of the wheat, whereby foodstuffs produced from the cereal grains have increased protein values. Cultures of the microorganisms in admixture with yeast may be used as a bread starter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.701,214, filed Feb. 13, 1985.

TECHNICAL FIELD

This invention relates to compositions and methods for improving thenutritive value of cereal based breads, to novel microbes useful in thefermentation of breads which thereby provide improvements in the bread'snutritive value, to grain and microbe mixtures, and to yeast and microbemixtures from which breads may be produced.

BACKGROUND

Bread produced from wheat flour is a major component of the human dietin many areas of the world, providing most of the required calories andproteins in the diet. In lesser developed countries, the caloriccontribution of bread to the diet may be as high as 80-90%. As much as64% of the daily protein intake in the developing countries is derivedfrom cereal grains from which bread is made. Flat breads, which are madeof cereal grains, are the most ancient of breads, and remain especiallypopular in the Middle East and Indian subcontinents. Many types of flatfermented breads, such as Egyptian baladi bread, are to be found aroundthe world. Standard characteristics include the flat shape, usually withan open, pocketed interior. The doughs are comprised of low proteinflour of high extraction, water, salt and leavening agent, either yeastor lactic acid-producing bacteria as a sour starter. The formula forBaladi bread dough consists of high extraction (82 to 88%) flour, watersalt, and 12 to 17% starter (fermented dough). Fermentation in the doughis initiated by the activity of wild bacteria and yeast which arepresent in the starter. For most flat breads the fermentation period isquite short, and is initiated by the activity of bacteria and yeast. Theheart of the baking process for all flat breads is the naturallyoccurring sourdough starter containing microbes. The sourdough starteris responsible for the improved leavening action, the bread's sour tasteand an extended shelf life.

Following the brief fermentation, the dough of most flat breads isdivided, flattened to a form resembling a pancake on a wooden boarddusted with wheat bran, and then proofed for another short period. Theflattened dough is then baked at high temperatures (400°-500° C.) for ashort time (1-3 min.). The flat piece of dough rises in the oven. Steamformation, rather than gas production, causes it to separate into twothin layers and form the characteristic pocket.

The formula and baking process for producing baladi, and other flatbreads are as much a part of tradition as the bread itself. Accordingly,one of the objects of the invention is to provide the means whereby theproduct can continue to be manufactured economically, in a mannercongruent with tradition, and also in such a way as to improve thenutritive value of flat bread. Although the conventional proceduredescribed above yields an excellent product, it is subject to certainnutritional disadvantages as outlined herein and a primary objective ofthe invention is to provide a procedure which alleviates thesedisadvantages.

Normal cereal grains, including the wheat from which most breads areproduced, are low in some of the essential amino acids, i.e., lysine,threonine, methionine, tryptophan and isoleucine, the so-called"limiting" amino acids. Such cereal grains can be considered low qualityprotein sources. Thus, cereal grain-based diets, prevalent in many areasof the world, may be deficient in some essential amino acids.

Research has proven that if humans lived primarily on cereal grains withno intake of animal protein, as is the case for people who have flatbread based diets, the protein received would be adequate if it were ofa quality comparable to animal protein. Thus, improving the nutritivevalue of fermented breads such as flat-breads, is of considerableimportance to lesser developed countries and other wheat importingnations. This is most effectively accomplished by increasing the lysinecontent of cereal protein. Further efforts may involve increasing thethreonine content thereof, as threonine is generally the next mostlimiting amino acid after lysine. Accordingly, it would be of tremendousimportance and benefit to produce a bread or wheat product with anincreased content of the limiting amino acids, thus raising thenutritional value of such products.

The nutritive value of the bread depends upon the protein level in theflour and on the balance of various amino acids that make up theprotein. Normal cereal grains, including wheat, are low in some of theessential amino acids; that is, lysine, threonine, methionine,tryptophan, and isoleucine, the so-called limiting amino acids. Thislimitation causes protein in the bread to be poorly utilized by thebody. Accordingly, it would be of tremendous importance and benefit toproduce a wheat bread with an increased content of limiting amino acids,thus raising the nutritional value of such products. In order for thehuman body to properly utilize protein, the essential amino acids mustbe available simultaneously and in the most advantageous proportions.The FAO (Food and Agriculture Organization) recommends a minimum lysineratio of 5.2% in protein as an ideal proportion of lysine for infants.Wheat protein generally has only about 50% of this ideal recommendedlevel. If lysine can be added to such wheat-based diets, a higher valueof wheat protein can be realized.

Such approaches as breeding and growing cereal grains for quality haveshown that increasing the nutritional value of cereal grains leads to adecreased yield and vice versa. Blending wheat flour with alternateprotein sources such as soybean meal or fish meal has been generallyunacceptable because of the cost of the protein source and tastepreference or consumers' acceptance.

The prior art teaches that various microorganisms and their mutantstrains have been used to produce lysine. For example, U.S. Pat. No.2,841,532 to Kita et al discloses the use of E. coli to produce lysine.U.S. Pat. No. 2,979,439 to Kinoshita et al teaches that lysine may beproduced from a mutant of Micrococcus glutamicus. U.S. Pat. No.3,524,797 to Boyd et al discloses a method wherein lysine is produced bycultivating a double mutant of Micrococcus glutamicus. In U.S. Pat. No.3,527,672 to Kubota et al, lysine is produced from mutant strains ofBrevibacterium lactofermentum. U.S. Pat. No. 3,756,916 to Leavittteaches specific methods for isolating a mutant strain of an aminoacid-producing microorganism, usually Brevibacterium glutamicus. U.S.Pat. No. 3,905,866 to Watanabe et al describes a process for theproduction of lysine by a mutant strain of Pseudomonas or Achromobacter.U.S. Pat. Nos. 4,275,157 to Tosaka et al and 4,411,997 to Schimazki etal teach the production of lysine by culturing mutants ofCorynebacterium or Brevibacterium.

None of these prior art teachings, however, teach or suggest theinoculation of lysine-excreting mutant strains into a bread or wheatproduct to enhance the lysine content thereof and thereby increase thenutritional value.

The prior art also discloses that it is known to use bacteria in theproduction of bread. For example, U.S. Pat. Nos. 3,734,743 and 3,891,773to Kline et al teach the use of Lactobacillus sanfrancisco in preparingsourdough breads. U.S. Pat. No. 3,963,835 to Gryczka teaches that wildtype Lactobacillus fermenti may be used to produce sourdough bread.However, to Applicants knowledge, there has not been any disclosure forimproving the nutritive value of fermented breads through the use oflysine-excreting microorganisms or use of lysine-excreting bacteria in abread or wheat products.

SUMMARY OF THE INVENTION

It is therefore one object of the invention to provide alysine-excreting microorganism to improve the nutritional value ofgrains and breads.

A further object of the present invention is to provide a pure cultureof the microorganism which may be used to improve the nutritional valueof bread.

A still further object of the invention is to provide a method forproducing the lysine-excreting microorganism.

It is even a further object of the invention to provide a wheat productenhanced in protein using a lysine-excreting microorganism.

A still further object of the invention is to provide a method forenhancing the protein quality of wheat that involves treating the wheatwith a lysine-excreting microorganism.

A further object is to provide a bread starter which comprises adominant, non-pathogenic active fermenting, non-biohazardous lactic acidbacteria combined with the proper level of baker's yeast for leavening.

A further object of the invention is to provide an enhanced proteinquality bread which has been treated with a lysine-excretingmicroorganism.

An additional object of the invention is to provide a method forenhancing the protein quality of bread which comprises treatment of thebread with a lysine-excreting microorganism.

Other objects and advantages of the invention will become apparent asthe description thereof proceeds.

In satisfaction of the foregoing objects, the present invention providesa novel microorganism having the identifying characteristics of thespecies Lactobacillus fermentum Lex⁺, methods for production thereof,and methods for the introduction of the microorganisms into wheat andbread products to provide said products with improved nutritive values.

This invention includes the method for spontaneously mutating andselecting the heterofermentative bacterium Lactobacillus fermentum,isolating the lysine-excreting strain L. fermentum Lex⁺ from the wildtype L. fermentum, forming a pure culture, optionally freeze-drying saidpure culture, and forming a sourdough starter comprising wheat flour,salt, water and the aforementioned pure culture of L. fermentum Lex⁺.This starter may be added during the fermentation period of the bread orwheat product to increase the nutritive value. Also provided are theresulting improved wheat and bread products treated with the culture.The invention also includes grains such as wheat which have been treatedwith culture, preferably in freeze-dried form. The invention alsoincludes a bread starter comprising the culture combined with yeast.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the drawings accompanying the applicationwherein:

FIG. 1 is a diagram showing the presumed lysine biosynthesis pathway inL. fermentum; and

FIG. 2a is a graph which illustrates the feedback-inhibition ofthreonine on lysine biosynthesis in L. fermentum;

FIG. 2b is a graph which illustrates the release of feedback-inhibitionof threonine on the lysine biosynthesis in L. fermentum;

FIG. 3 is a graph showing the lysine level as affected by degree offlour extractions using the bacterium of the invention; and

FIG. 4 is a graph showing the effects of bacterial inoculation dose onthe development time of baladi bread dough.

DESCRIPTION OF PREFERRED EMBODIMENTS

As discussed, the present invention relates to mutant microorganismscapable of excreting excessive lysine, methods for selecting andisolating these microorganisms and wheat and bread products inoculatedwith these mutant organisms, which products have increased nutritionalvalue due to their higher lysine content. In a preferred embodiment,these mutant microorganisms are strains of Lactobacillus fermentum, ahetero-fermentative lactobacillus. The invention is concernedspecifically with an organism having the identifying characteristics ofLactobacillus fermentum Lex⁺ strains M11 and M14 which are capable ofproducing an excess amount of amino acids, lysine in particular.Further, the invention is concerned with uses of these bacteria inincreasing the nutritive values of grains and breads or other foodsproduced therefrom.

In order to place the invention in proper focus, it is necessary tooutline the nutritional limitations of wheat used in bread production.The quality of consumed protein in the nutrition of a person is of equalor greater importance than the quantity. In order for the human body toproperly utilize protein, the essential amino acids must be availablesimultaneously in the most advantageous proportions. If one of theessential amino acids is missing (limiting), body utilization of otheramino acids is "wasted" in that they are used up for energy rather thanfor vital regulatory or protein building processes.

High quality proteins upon digestion provide balanced supplies of thehuman's essential amino acids and can subsequently be absorbed asprecursors for body protein formation. Because it is well establishedthat the normal cereal grains, including wheat, are low in some of theessential amino acids; i.e., lysine, methionine, threonine, tryptophanand isoleucine, such cereal grains can be considered as low qualityprotein sources.

Lysine is the initial amino acid normally limiting in cereal grains.Assuming that the UN-FAO (Food and Agriculture Organization) ratio of5.2% lysine in the protein comprises the ideal proportion of lysine forinfants, wheat protein generally has only about 50% of the idealrecommended level. According to FAO data, cereal grains comprise 65-66%of the calorie supply for people of developing countries, whereas animalproducts provide merely 6% of the calories. Information from FAO alsoreveals that in the Middle East, plant sources provide 80% of the dailyprotein supply, with animal source foods supplying only 20%. Thus, giventhe previous information, the average diet in developing countries isprimarily comprised of low value proteins that are lysine deficient.

If lysine can be added to such wheat based diets, a higher value forwheat protein can be attached. For example, egg protein is consideredthe food with the most ideal proportion of amino acids, with anutritional value of 93 on a 100 point scale. If wheat flour issupplemented with 0.10% lysine, its protein value can be increased from35 to 55. Even smaller increases in lysine content can make asignificant improvement in protein value.

Another test of protein quality is the Protein Efficiency Ratio (PER).It represents the amount of weight gained in proportion to the amount ofprotein fed. An example of this test, using wheat, provides a PER of0.93. By adding the same 0.10% of lysine, the PER is raised to 1.45, anincrease of one and one-half times the original.

Research has shown that if humans lived on primarily cereal grains suchas flat breads, with no intake of animal protein, the amount of proteinconsumed would be adequate if it were of a quality comparable to animalproteins. Improvement of the nutritive value of cereal based breads, theprimary objective of the invention, is of considerable importance tolesser developed countries as well as other wheat importing nations.This is most effectively accomplished by first increasing lysine.Further nutritional improvement in fermented breads would involveincreasing threonine, the next most limiting amino acid.

This nutritional improvement of bread can be achieved by utilizingseveral techniques, including that presented in this invention. It isimportant that these various different means, aside from the invention,and their limitations be understood for complete comprehension of theinvention's significance.

The aforementioned wheat quality deficiencies can be rectified bybreeding and growing cereal grains for quality. However, the limitationsof breeding wheat toward improved nutritional value are many. Plantbreeding comprises a selection for the traditional problems of highyield, drought resistance, pest resistance, etc., with each componentadding an ever increasing level of difficulty, and the addition ofnutritional quality only further increases the burden on the breeder. Ithas been shown that increasing the nutritional value of cereal grainsleads to a decreased yield and vice versa. This inverse correlationbetween breeding yield and nutrition is due to a forced redeployment ofplant energy from carbohydrate storage to protein storage. In addition,wheat breeding is an exceedingly slow process, with the creation of anew variety and its introduction into the field taking as long as tenyears. In addition, the plant breeding process is subject to suchadditional hazards as adverse climate, high production cost, anddependence on fertilizer. Ultimately, breeding desirable nutritionaltraits into wheat by any methodology is a multi-faceted problem.

Blending wheat flour with alternate protein sources such as soybeansalso improves the bread's nutritional balance. The problems with thismethod are the cost of the protein source and taste preference oracceptance by the consumer. Another complication is that part of thelysine added to bread in pure form (1-lysine-HCl) or in bound form isdestroyed during baking. The percent loss during baking ranges between12 and 21%.

With the biotechnical approach of this invention, the protein quality ofany bread can be improved or supplemented by fermenting the doughutilizing special strains of bacteria capable of producing high levelsof amino acids that are low in the cereal flour, particularly lysine.This supplementation by fermentation is the method encompassed by thepresent invention. The invention thus involves mutants of the bacteriumLactobacillus fermentum (strains M11 and M14 Lex⁺). These bacteria,indigenous to Egypt, were isolated from native sourdough starter, anddeveloped as novel strains according to the novel procedures set forthherein.

The bacteria L. fermentum Lex⁺ strains M11 and M14 were produced astaught herein to provide an advantage over starter bacteria of the wildtype, due to their increased ability to excrete lysine. In oneembodiment, the invention includes procedures to isolate, mutate,select, and then reintroduce the L. fermentum Lex⁺ into native sourdoughstarter. When this bacteria is added to the starter, bread such as flatbread can be produced in the conventional manner with the advantage ofincreased lysine levels, which subsequently improve the bread'snutritive value.

This biotechnical technique is superior for several reasons to alternatemeans presented for nutritionally improving bread. First, it iseffective regardless of the initial quality of the flour. It can beutilized in addition to research findings concerning other nutritionalimprovement strategies. As pointed out, it is compatible with theeconomy, tradition and time constraints involved in producing anutritionally improving bread, such as flat bread, native to developingcountries.

Preparing the starter with the improved microbes is a relatively simpleand inexpensive process. It also permits developing countries tocontinue importing the relatively inexpensive wheat necessitated bytheir limited economies. This method also allows for a great deal offlexibility because countries or individuals have the choice of whetheror not to use the starter enhanced by L. fermentum Lex⁺. The productionof new strains of bacteria can proceed far more rapidly and with greatergenetic flexibility than can the production of new wheat cultivars.Thus, the present invention is concerned with the organism Lactobacillusfermentum Lex⁺, strains M11 and M14, and the process of improving thenutritive values of grains and cereal based bread. As noted above, theinvention is useful in providing sourdough starter and resulting breadwith increased nutritive values. In a further aspect, the inventionprovides procedures and compositions by which the nutritive value ofwheat or other grain is increased. Thus, the novel bacterium of theinvention may be added to the wheat or other grain in bulk. Thereafter,bread produced from the treated wheat will have the increased nutritivecontent.

Preferred species used in this invention are Lactobacillus fermentumLex⁺ strain M11, ATCC 39910 and Lactobacillus fermentum Lex⁺ strain M14,ATCC 39911. A culture of each of these preferred species has been placedon deposit with the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852, and the accession numbers set forth abovehave been assigned thereto. In each case, the deposit is restricteduntil such time as a patent has been issued disclosing the abovedeposits, except that access is available under 37 CFR 1.14 and 35 USC122. After issuance of the patent, all restrictions on availability ofthe deposited cultures to the public will be irrevocably removed. Thedeposits will be maintained for a period of 30 years from the depositdate, and accordingly, the cultures will be permanently available to thepublic after issuance of the patent.

Hetero-fermentative lactobacilli utilize the hexose prophosphate shunt(Embden-Meyerhof pathway, mixed acid class) and produce from each moleof glucose fermented one mole each of lactic acid, ethanol and CO₂. Anindigenous Egyptian sourdough starter (Soltani) was obtained from theSeed Technology Lab at the Agricultural Research Center in Giza, Egypt.The predominant fermenting component of such a starter includes a groupof bacteria (lactobacilli) that produce lactic acid (the sourness) andimperfect yeasts that produce CO₂ which slightly leavens the bread.Several isolations were made from the Soltani starter using lysine assaymedium with the addition of maltose (0.1% w/v). With hetero-fermentativelactobacilli, both CO₂ and lactic acid are produced and the acid productis 50% less than under homo-fermentative conditions, wherein two molesof lactic acid are produced from each mole of glucose. The slowerincrease in acidity permits fermentation, and thus lysine production toproceed for a longer period. Species of the hetero- fermentativebacteria, Lactobacillus fermentum were used for selection and mutationin this invention. The L. fermentum has the following description: Grampositive rod, non-motile, catalase negative, produces acid and gas fromglucose and gluconate, can ferment arabinose, galactose, lactose,mannose, and xylose but not cellobiose and trehalose, grows at 45° C.;not at 15° C. (Bergey's Manual, 1974).

Wild types of L. fermentum obtained from Egypt do not produce detectableexcess amino acids. They merely produce sufficient amino acids for theirown use. Thus, in order to obtain strains which have the identifyingcharacteristics of M11, ATCC 39910 and M14, ATCC 39911, of L. fermentumLex+ which are capable of excreting excess amino acids, particularlylysine, it was necessary to select repeatedly.

The preferred species were selected as regulatory mutants of aparticular system of lysine synthesis, said systems being demonstratedby FIGS. 1 and 2a accompanying the application. According to theregulatory systems, one aspartokinase is subject to multivalent feedbackinhibition by threonine and lysine (FIG. 2a), and one homoserinedehydrogenase is subject to feedback inhibition by threonine andrepression by methionine (FIG. 1). Accordingly, these regulatory systemswere used to isolate regulatory mutants from L. fermentum by isolationof L-lysine analog-resistant mutants.

According to the invention, the selection procedures involve exposingthe bacteria to sequentially higher levels of the following amino acidanalogs, first singly and then in combination: 5-S-amino ethyl cysteine,gamma-hydroxylysine, lysine hydroxymate and cyclohexylalanine. Theresulting mutants synthesize aspartokinase insensitive to multivalentfeedback inhibition by lysine or threonine to increase the production oflysine from aspartate. To maximize the flow from aspartate to lysine,further selections are carried out and mutants with homoserinedehydrogenase insensitive to feedback inhibition by threonine andrepression by methionine are isolated. Examples of such mutants includebut are not limited to Met⁻, Thr⁻, Ile⁻, Lex⁺ and Eth^(R). The mutationselection cycle is repeated as many as ten to fifteen times to increasethe genetic variability in the microorganisms. In addition tobiochemical marker systems, antibiotic marking is also used inidentification and selection of the mutants.

Measurement of the amino acid excretions by the mutants was made by across-feeding method and thin layer chromatography with modified mobilephase (n-propanol, 58 parts; NH₄ OH, 27 parts; and H₂ O, 15 parts),which permitted several thousand isolates a day to be screened. Thestrains' ability to excrete lysine was manifested by inoculation into30% (W/V) wheat flour/water extract. After over-night incubation, thelysine bioassay using Leuconostoc mesenteroides (Pediococcus cerevisiae)(ATCC 8043) was applied to select the highest lysine excretors. Thepreferred species of this invention (strains M11 and M14 of L. fermentumLex⁺) proved to have excreted lysine in the highest amounts. As notedabove, these have been deposited with ATCC, under Accession Numbers39910 and 39911, respectively. In its broadest aspect, this inventioncovers any microorganism which has been mutated and isolated from aproven biosynthetic pathway and which excretes an excess amount oflysine and which has the identifying characteristics of either M11, ATCC39910, or M14, ATCC 39911.

In conducting the selection process, pure strains are initially grown ina suitable broth and then washed with a buffer solution such aspotassium phosphate. The resulting cells are then suspended in a lysineassay broth and incubated. Incubation conditions are generally for 14 to20 hours at 34° to 38° C., under which conditions only the resistantcells will continue to grow, and these can be seen to increase to thepoint of turbidity, i.e., greater than 10⁷ cells per milliliter. Aserial transfer is then made to a fresh container of the same broth, soas to eliminate "escape" cells. The lysine treatment, incubation andgrowth of resistant cells followed by separation, is repeated severaltimes with sequentially higher analog concentrations. Theseconcentrations begin at 1000 and increase up to 100,000 ppm analog. Theanalogs used are 5-S-amino ethylcysteine, gamma-hydroxylysine, andcyclohexylalanine. From the highest concentration, the cultures arediluted and plated onto indicator plates. The plates should containlysine assay media seeded with a lysine requiring bacterium. Thebacteria producing the largest zones of indicator bacteria are streakedto purity and then single colonies are bioassayed. Repeating these stepswith several analogs individually and finally in combination willprovide mutant strains producing the maximum concentration of lysine.The resultant strains selected by this procedure have the capacity toexcrete significantly higher amounts of lysine than is excreted by thewildtype or natural strain of this microorganism.

In a second aspect of the present invention, pure cultures of freezedried L. fermentum Lex+ are provided. In this aspect of the invention,the bacteria which are isolated in accordance with the method of theinvention, were found to grow well on MRS broth at 37° C. (98.6° F.) for12-16 hours. The mass cells were separated by centrifugation and washedthree times with a sterile saline solution and then resuspended in 0.1%w/v, sodium alginate by the method disclosed in Japanese Pat. No.15/43098, 1977, the disclosure of this Japanese Pat. No. 15/43098 beingincorporated herein by reference. The suspension was freeze-dried,packed in closed polyethylene bags and stored dry at room temperature.By using this method, it was demonstrated that the viability offreeze-dried bacteria could be maintained indefinitely. Further, it wasfound that lysine excretion efficiency is not adversely affected by thefreeze-drying process. This embodiment provides the microorganism in aform suitable for commercial use.

In a third aspect of the present invention, cereal grains treated withthe microorganisms of the invention are provided, which have enhancedprotein quality. In a further embodiment of the invention, enhancedprotein quality wheat is used to produce fermented breads with enhancednutritive value. Enhanced nutritional value in wheat products isparticularly advantageous agriculturally in developing countries wherecereal grains comprise 65-66% of the caloric intake of the people ofthose countries.

An important aspect of the invention resides in treatment of cerealgrains with the microorganisms of the invention, as indicated above. Thepreferred cereal grain to be treated is wheat, since bread is made fromwheat as described herein. Thus, in a preferred embodiment of theinvention, cereal grains such as wheat are treated in bulk withmicroorganisms which have the characteristics of those described herein.The microorganisms are preferably applied in freeze-dried form, whichmay be produced as indicated above. A sufficient amount of the freezedried organism or culture should be added to the wheat in bulk toprovide the necessary enhanced nutritive value. The amount offreeze-dried culture to be added to the bulk cereal grain will usuallybe sufficient to provide a bacterial count to the wheat of about 10³ to10⁸ cells per gram of wheat, or about 0.1 to 100 ppm for wheat treatedin bulk. Preferably, there is present sufficient culture to be presentin the range of about 10⁶ to 10⁸ cells/gram flour.

In this aspect of the invention, the wheat or other cereal grain can betreated with the freeze dried culture in bulk prior to shipping.Thereafter, when the wheat is used to make bread, the culture will bepresent to increase the nutritive level of the resulting product, suchas bread.

In another embodiment, there is provided a method for creating a startermaterial useful for enhancing the protein quality of wheat and bread. Tocreate the starter in one aspect, a basic formula of soft wheat flour of82% extraction, 100 parts; table salt, 1.2 parts; water, 70 parts (v/w);and freeze-dried bacterial cells were used. The water proportion wasvaried within certain limits (65% v/w often used). The starter was madefrom a pure culture of L. fermentum Lex⁺ (M11 or M14) as provided by theinvention or a portion of developed starter obtained from a previousbatch fermented with L. fermentum Lex⁺ (ATCC 39910 or ATCC 39911).

The above innoculum was added in such doses as to provide a maximumlysine level during the dough fermentation period (10-12 hrs) withregard to the level of sourness (lactic acid) accepted by the breadconsumers. Dose experiments, for example, showed that starter additionsat a zero (make-up) time from 10⁶ to 10⁸ bacterial cells/gram flour weresufficient to produce a high nutritive value dough. During thedevelopmental period, at room temperature, i.e., 25°-30° C. (77°-86°F.), the bacterial cell mass increased approximately 10-12 times, the pHdropped to 4.1-3.8 and the total lysine content reached 3.22%,considerably above the normal content of 2.87%. Portions of thedeveloped starter (1.2×10⁸ -1.2×10⁹ cells/gram flour dry base) could berefrigerated (4° C.) for use in the next batch. The starter may berecycled as long as the batches are prepared daily or can berefrigerated. If the starter shows any undesirable signs, such asspoiling or decrease in strength, the preparation of new starter usingfreeze-dried bacterial cells is preferable. In order to ensure highquality bread under the conditions prevailing in developing countries,the starter should not be recycled more than 7-10 times. Otherwise,continuous amendment of wheat flour by the proper number of thefreeze-dried bacteria should be taken into consideration.

In a further embodiment of the invention, there is provided a method forbatch- fermenting dough into bread simulating the commercial practice inEgypt. The primary component of the bread dough was a soft wheat flourof 82% extraction and 11.2-11.8% protein (air dry base). The doughingredients included 100 parts by weight flour, 1.2 parts sodiumchloride, variable amounts of water, and starter from either freezedried bacterial cells (10⁶ -10⁸ cells/g flour), as provided in anotheraspect of this invention, or a portion of the last fermentation batchprovided by the bacteria of the present invention; i.e., 10-12 parts.

According to this aspect of the invention, the ingredients were mixedtogether for 5 minutes and then allowed to ferment for 8-10 hours at25°-30° C. (77°-86° F.) at over 90% relative humidity. The dough wasdivided and formed into loaves which were 6 mm thick, measured 15-20 cmin diameter, and 65 g. in weight. The molded loaves of dough weretransferred onto trays that had been dusted with a thin layer of bran.They were then placed in the proofing cabinet for 1 hour at 30° C. (86°F.) and 90% relative humidity. Samples were taken from the fermenteddough and some were placed in the refrigerator for the next batchfermentation.

The amount of water needed depended on the water absorption of theflour, which was variable. Larger amounts of water tended to make thedough very slack, requiring special handling techniques. The elevatedwater levels, however, developed a highly nutritive dough. Using softwheat flour at 82% extraction at levels of 60-75% water, the highestlevels of free lysine were obtained. Under such conditions, the lysinebioassay technique was used on the dough fermented with both the wildtype bacteria, and the modified L. fermentum Lex+ (ATCC 39910 and 39911)bacteria. The lysine bioassay technique revealed that a significantamount of lysine had been excreted in the modified system. The lysineexcretions from the system of the invention reached a level of 3.2-4.7%of the total protein content as compared to 2.0-2.5% in dough developedfrom the wild type L. fermentum (p<0.001).

The proofed dough was transferred onto a wooden peel which was used toslide the dough into the oven. The dough was baked in a carousel oven ata temperature of 354° C. (670° F.) for 2-2.5 min. Following baking,samples from the baked bread were freeze-dried and then ground for aminoacid analysis.

In a still further embodiment of the invention, there is provided yeastand microbe mixtures as bread starters. This embodiment provides astarter mixture which will consistently provide a bread of high proteincontent, thus providing standardization in the bread making process.

Standardization of any system is considered the first step formanipulation and improvement of the system and determine any significantcontribution of the suggested improvement. However, there has not beenany standardization known for the Baladi bread starter, especially themicrobial components. The lack of information about the identity ofthese microbial components has prevented work to improve the quality ofBaladi bread. By the methods and products of the present invention,standardization of the starter is now possible through the utilizationof the dominant, non-pathogenic, active fermenting, non-biohazardouslactic acid bacteria of the invention combined with the proper level ofbaker's yeast for leavening.

Traditional recycling of Baladi starters gives unpredictable results andcan produce health hazards. Microbial contamination of the starter maytake place during handling and processing of dough by experiment and/orpersonnel. In private bakeries, especially in villages and small townsor whenever the environment is polluted, there are no attempts to cleanor sanitize the equipment to reduce such contamination. Continued use ofthe starter technique over longer periods may increase the contaminationof the starter by other microorganisms.

It has been discovered according to the present invention that when themicrobes of the invention are used to initiate the starter, the breadcan be uniformly produced in the conventional manner. However, becauseof its increased ability to excrete lysine, the resulting bread willhave the advantage of increased lysine levels and will be free ofcontamination. The result will be improved nutritive value of the bread.

This technique has proven to be superior since it is effectiveregardless of the initial quality of the flour, and it is compatiblewith the economy, tradition, and time constraints involved in producingBaladi bread.

According to the present invention, these improved results can beobtained using a mixture of 10 to 40 parts of the lactobacilli starterfermented with 10⁶ cells/gram of flour and about 175-275 mg of dry yeastper kg of flour. Preferably about 20-30 parts of the lactobacilli pergram of flour are used with 200-250 mg of yeast per kg of flour. Thepackaged mixture of yeast and microbe would preferably contain a cellcount of yeast to microbe of about 1:100 up to 1:1000.

In a most preferred aspect, the Lactobacillus fermentum can be suppliedin a freeze-dried form in packages sealed carefully to protect theviability of the bacteria. Such packages will need little special carein handling or storage. Further, in the automatic or semi-automaticsystems, the Baladi bread formula usually contains 2.5 to 3 g dryyeast/kg flour which is equivalent to 2.5 to 3.0 kg/ton. UtilizingLactobacillus fermentum of the invention requires only 0.25 to 0.30 gdry yeast/kg flour which is equivalent to 250 to 300 g dry yeast/tonflour (about 10% of the original amount). The improved Baladi bread willhave 20.1% higher lysine content than the conventional, which willprovide 20.1% more protein for the people.

Specific examples of the present invention will now be set forth. It isunderstood that these examples are merely illustrative, and are in noway to be interpreted as limiting the scope of the invention. In theexamples and throughout the application, parts are by weight unlessotherwise indicated. In this specification, reference to amino acidsmeans 1-amino acids.

EXAMPLE 1

This example describes the selection process for obtaining the mutatedstrains of the Lactobacillus fermentum Lex⁺ microorganisms identified asATCC 39910 and 39911. An indigenous Egyptian sourdough starter (Soltani)was obtained from the Seed Technology Lab at the Agricultural ResearchCenter in Giza, Egypt. The fermenting component of this starter includeda group of Lactobacilli. Species of the heterofermentative bacteria,Lactobacillus fermentum, were used for selection and mutation. The L.fermentum is a Gram positive rod, non-motile, catalase negativebacteria. Pure strains of this species were grown for 16 hours in MRSbroth. They were then washed three times with 0.05 molar potassiumphosphate buffer at a pH of 6.8. The washed cells were then resuspendedin 0.3 milliliters of lysine assay broth, which contained 500 ppm oflysine analog. This suspension was then incubated at 35° C. for 16hours. During incubation, only the resistant cells continued growing,and these could be seen to increase to the point of turbidity. Turbidityoccurs at a concentration of greater than 10.sup. 7 cells permilliliter. A serial transfer was then made to a new tube containing thesame broth, but eliminating the "escape" cells. The preceding steps ofwashing, incubating, growing and transferring were then repeated 2 to 4times with sequentially higher analog concentrations, i.e., 1,000,2,000, 5,000, 10,000, 30,000, 50,000 and 100,000 ppm analogs. Generally,the analogs in the order that they were used were5-S-amino-ethyl-cysteine, then gamma-hydroxylysine, and thencyclohexylalanine. Combinations of the first two analogs were made, thenall three were combined.

From the highest concentration, the cultures were diluted and platedonto Difco lysine assay medium to obtain isolated colonies. Singlecolonies were chosen at random and patched onto indicator platescontaining lysine assay agar seeded (10⁶ /ml) with lysine auxotrophbacteria, Leuconostoc mesenteroides (Pediococcus cerevisiae) ATCC 8043.Colonies which produced the largest zones of indicator bacteria werestreaked to purity, and single colonies were bioassayed in 5 milliliterlysine assay broth tubes. The steps of diluting and plating ontoindicator plates followed by streaking to purity and bioassaying wererepeated using three different analogs individually, and finally incombination, to provide a maximum total concentration of 100,000 ppmanalog. The recovered bacteria were identified as M11 and M14, which aredescribed herein as ATCC 39910 and ATCC 39911. These were the bacteriaused in the following further examples.

EXAMPLE 2

A Baladi bread production trial was conducted using the followingformulation:

    ______________________________________                                        Soft wheat flour 82% ext.                                                                           100    parts                                            Water                 70     parts                                            Sodium chloride       1.2    parts                                            Bacterial starter     10.sup.8                                                                             cells/g flour                                    (freeze-dried strain ATCC 39910)                                              ______________________________________                                    

The dough ingredients and starter were mixed for three minutes,fermented for eight hours; then 11/2 hours proofing, baked for 21/2 min.at 354° C. (670° F.), cooled at room temperature for two hours,freeze-dried, then ground with a flour mill. Samples from the proofeddough were also freeze-dried. Non-fermented (no bacteria) and wildtypebacteria doughs were used as controls. Samples were analyzed for aminoacids by ion-exchange chromatography.

The following Table 1 sets forth the results of the amino acid analyses.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Content* of Baladi Bread                                                  Selected                                                                      Mutant        Wildtype                                                        ATCC 39910    Bacteria      Non-fermented                              Amino Acid                                                                             dough   bread   dough bread dough bread                              ______________________________________                                        Aspartic Acid                                                                          5.69    5.59    5.56  5.33  5.57  5.31                               Threonine                                                                              2.97    3.01    3.03  3.09  3.04  3.08                               Serine   4.21    4.22    4.38  4.73  4.39  4.71                               Glutamic 31.02   31.07   31.23 31.44 31.00 31.45                              Acid                                                                          Proline  10.3    10.76   10.35 10.57 10.64 10.37                              Glycine  4.37    4.30    4.29  4.30  4.22  4.37                               Alanine  3.80    3.79    3.70  3.78  3.72  3.77                               Cystine  1.65    1.64    1.68  1.63  1.60  1.63                               Valine   4.95    4.91    4.80  4.73  4.81  4.80                               Methionine                                                                             1.73    1.72    1.77  1.80  1.77  1.71                               Isoleucine                                                                             3.71    3.70    3.70  3.69  3.72  3.68                               Leucine  7.10    7.14    7.24  7.22  7.18  7.11                               Tyrosine 2.81    2.67    2.78  2.75  2.87  2.74                               Phenylalanine                                                                          4.87    4.91    4.97  4.98  4.98  5.06                               Histidine                                                                              2.56    2.58    2.61  2.58  2.62  2.57                               Lysine   3.22    3.10    2.95  2.58  2.87  2.74                               Arginine 5.03    4.91    4.97  4.81  4.98  4.88                               ______________________________________                                         *Analyzed by Ionexchange chromatography at Experiment Station Chemistry       Laboratory, Univ. Missouri  Columbia, Dr. Charles Gherke. Values represen     average of three replications as percent of amino acids per total amount      of amino acids present.                                                  

As will be noted from Table 1, the amino acid analysis of the Baladibread mixture fermented with the selected mutant L-fermentum Lex⁺(strain ATCC 39910) or its wildtype and the control dough, both beforeand after baking, showed no significant differences in the content ofamino acids other than lysine. Lysine, however, was significantly higherin the doughs or breads (8.5 and 21.71% respectively) fermented with theselected mutant than those fermented with the wildtype, (P<0.001) orcontrol. During baking, the conventional wildtype fermented doughdecreased in lysine by 14.1% of the total lysine content. In contrast,the dough fermented with the lysine excreting mutant decreased only 4.1%of the total lysine content. The slight increase in the lysine contentin the conventional system over the control may be explained byexperimental error and/or due to the mass of bacterial innoculum usedfor fermentation.

Loss of lysine during baking is a common phenomenon, wherein compoundsare formed which destroy or bind lysine into unavailable forms.Monomolecular destruction, strong Maillard reaction and dextrinizationof starch at high baking temperatures have been reported as causativefactors. The explanation of this apparent protection of lysine duringbaking by the mutant fermentors of the invention, as compared to thewildtype, is not clear.

EXAMPLE 3

This experiment illustrates the effects of various dough water/flourratios on bacterial lysine production in the dough. A series of doughswere prepared with various levels of water as shown in Table 2 andfree-lysine content was determined as shown in Example 3. Both M11 andM14 bacteria were used and the wild type bacteria was used as a control.

                                      TABLE 2                                     __________________________________________________________________________    Effects of Water Levels Used for Mixing Dough on Lysine                       Excretion in the Developed Dough                                                        pH               Free-Lysine Content                                Water     Wild-                                                                             ATCC                                                                              ATCC     Wild-                                                                             ATCC                                                                              ATCC                                       Levels                                                                             Control                                                                            type                                                                              39910                                                                             39911                                                                             Control                                                                            type                                                                              39910                                                                             39911                                      __________________________________________________________________________    55 parts                                                                           6.36 3.80                                                                              3.93                                                                              3.94                                                                              0.5  2.45                                                                              3.55                                                                              3.68                                       65 parts                                                                           6.31 3.78                                                                              3.88                                                                              3.82                                                                              0.57 2.48                                                                              4.06                                                                              4.74                                       75 parts                                                                           6.30 3.77                                                                              3.81                                                                              3.79                                                                              0.81 1.98                                                                              3.28                                                                              4.01                                       85 parts                                                                           6.30 3.93                                                                              3.81                                                                              3.76                                                                              0.82 2.24                                                                              3.31                                                                              3.85                                       LSD                           0.27 (P 0.00105)                                __________________________________________________________________________

In this experiment, conventional bacteria may have caused a release ofsome of the bound lysine to the free form. Increasing the proportions ofwater in the dough mix had no significant effects on such activity.Using the new bacterium increased the amount of free-lysine contentsignificantly, presumably by releasing some of the bound lysine as wellas by lysine excretion. These effects account for increases in lysineover the wildtype ranging from 44-63% and 50-100% for the mutants ATCC39910 and ATCC 39911 respectively. Dough mixtures containing between60-75 parts water/100 parts flour (v/w) were shown to be the optimumratio for bacteria to grow and excrete a considerable amount of lysine.

EXAMPLE 4 Effects of Bacterial Inoculum Dose on Fermentation

This experiment illustrates the effect of a series of doses offreeze-dried bacterial cells on quality of the fermented dough. A seriesof doughs was prepared using the formulation described in Example 2 withthe addition of the bacterial doses shown in Table 3 as follows. A doughcontaining 0 cells/g flour served as a control. Cross-feeding bioassaysfor lysine using Leuconostoc mesenteroides (Pediococcus cerevisiae) ATCC8043 made on the water extracts of the fermented dough showed that 10⁶-10⁸ cell/g flour were required to obtain significant increases infree-lysine content.

                  TABLE 3                                                         ______________________________________                                        Effects of Modified Bacterial Dose on the Dough                               Nutritive Values (Free-lysine percent) of the Baladi                          Bread Fermented Dough                                                         Zero Time           Developed Dough after 10 Hrs.                             Dose Cells/g   pH       Cells/g pH     Free-lysine                            ______________________________________                                        1    3.97 × 10.sup.8                                                                   6.44       79 × 10.sup.8                                                                 3.80   4.97                                   2    5.68 × 10.sup.7                                                                   6.42      9.6 × 10.sup.8                                                                 4.20   3.26                                   3    5.68 × 10.sup.6                                                                   6.43     0.88 × 10.sup.8                                                                 5.16   2.25                                   4    5.68 × 10.sup.5                                                                   6.44     0.10 × 10.sup.8                                                                 6.12   0.06                                   5    5.68 × 10.sup.4                                                                   6.45     0.07 × 10.sup.7                                                                 6.24   0.06                                   6    5.68 × 10.sup.3                                                                   6.44     0.08 × 10.sup.6                                                                 6.48   0.07                                   7    0         6.50     0       6.48   0.004                                  LSD                      (P 0.0012)0.52                                       ______________________________________                                         *Bioassay method, percent total protein content; 11.2-11.8%.             

The degree of flour extraction or milling is an apparent factor inlysine production in the dough (FIG. 4). Using the higher flourextractions, 100% of the original wheat resulted in higher lysinecontent, compared with 72% extraction flour. Apparently the bran contentof the whole grain, 100% extraction, improved the growth of thesebacteria. This table indicates that about 10⁶ cells/g is a preferredlevel to attain for significant increased lysine levels.

In cases using the lower doses, it is preferable to ferment the doughfor lengthier periods to get significantly higher levels of lysine andacid. The bread sourness (acid) must be increased to an acceptable levelas evidenced by taste tests.

The time course study of fermentation (FIG. 3) shows an increase in freelysine concommitant with a decrease in pH.

EXAMPLE 5 Nutritive Analysis of Fermented Breads

This experiment tested the bioavailability of the proteins in the bakedBaladi bread fermented with the selected mutant L. fermentum Lex+(strain ATCC 39910) or the wildtype (WT). Baladi bread mix(non-fermented) and casein diets were used as controls. The dietcomposition is presented in Table 4 below using rats as control animals.

                  TABLE 4                                                         ______________________________________                                        Content of Rat Diets in Percents                                                           B.B. diet                                                                            Casein Control                                            ______________________________________                                        Baladi bread   88.00    --                                                    Corn starch    2.75     75.40                                                 Casein         --       11.85                                                 Vitamin mix    1.00     1.00                                                  Mineral mix    2.00     2.00                                                  Cellulose      1.50     1.50                                                  CaCO.sub.3     .75      .75                                                   Corn oil       4.00     4.00                                                  ______________________________________                                    

In conducting this experiment, weaning male Sprague-Dawley rats werehoused in cages with wire floors with feed and water provided (adlibitum). Ten rats were assigned to each test and diet treatments werestratified by row and tier to minimize cage location effects.

Table 5 summarizes the results obtained comparing Protein EfficiencyRatio (PER), body weight gain, and feed conversion rates for a 28 dayperiod. The conventional bacteria fermentation followed with high bakingtemperatures caused severe damage to the flour protein quality. Therewas a 17.2% (P<0.030) lower PER and 16.6% (p<0.02) higher conversionrate recorded in the conventional system as compared to non-fermentedflour. As shown in Table 5, this decrease in biological measures,resulted from the joint effects of the lysine destruction under highbaking temperature and of possible Maillard reaction which tended todecrease the bioavailability of lysine. In the growth comparison, theeffect was even more evident. Growth decreased by 27.5% (P<0.07) fromthe control. The selected mutant L. fermentum Lex+ strains (ATCC 39910AND 39911) successfully increased the nutritive value of the Baladibread (P<0.005). The excellent growth of the rats fed the casein dietindicated that the rats used in this experiment were normal, healthy andof good quality for testing problem quality. The new bacteriummanifested this improvement through the lysine excretion (22.7% higherthan conventional system; P<0.001). Apparently some kind of protectivemechanism decreased the amount of the destroyed or unavailable lysineduring baking at high temperatures (only 4.1% destruction in the newbread compared to 14.1% in conventionally fermented bread).

                  TABLE 5                                                         ______________________________________                                        Growth of Rats on Baladi Bread Basic Diets                                    G.                                                                            Gain       %*     PER       %**  Conv. Rate                                                                            %***                                 ______________________________________                                        A. Flour                                                                               37.8   27    1.53 + .20                                                                             17.7                                                                              6.98 + 1.2                                                                            -16                                control                                                                       B. Bread                                                                               29.7  0      1.30 + .25                                                                            0    8.36 + 2.0                                                                            0                                  Wild Type                                                                     C. Bread                                                                               40.4   36    1.62 + .14                                                                             24.6                                                                              6.42 + .06                                                                            -23                                Mutant                                                                        D. Casein                                                                             148.1  399    3.19 + .14                                                                            145.4                                                                              3.11 + 0.1                                                                            -62                                Control                                                                       p-Values                                                                      A × B × C                                                                       <0.05     <0.01     <0.02                                       A × B   <0.07     <0.036    <0.02                                       A × C   <0.5      <0.30     <0.35                                       B × C   <0.02     <0.005    <0.002                                      ______________________________________                                         *The growth gains over the wild type.                                         **The PER (Protein Efficiency Ratio) improvement over the wild type.          ***The conversion rates comparing to the wild type.                      

EXAMPLE 6

This experiment was conducted to determine the optimum bacterial dose tobe used for preparation of the starter for Baladi bread. Table 6 showsthe results which confirm that the best potential starter is preparedusing 10⁶ to 10⁷ cells/g flour (dry base).

The starter caused up to 71% leavening in the dough mix, decreased thepH to 4.79, and increased the amount of lysine by bioassay. Using either20% or 30% starter in the dough mix showed little differences in thequality of the fermented dough.

                                      TABLE 6                                     __________________________________________________________________________    Effect of Bacterial Dose in the Starter on the Quality                        of The Fermented Baladi Bread Dough.                                                                   Free                                                 Bacterial                                                                           Starter                                                                            Bacterial count (CFU/g) (2)                                                                 Lysine                                               dose (cells/                                                                        Level (1)                                                                          at mixing                                                                            After 3                                                                              by      Leavening                                    g flour)                                                                            (%)  time   hours  bioassay                                                                           pH %                                            __________________________________________________________________________    10.sup.4                                                                            20%  4.82 × 10.sup.6                                                                1.1 × 10.sup.8                                                                 252.4                                                                              6.32                                                                             7.2                                                30%  7.59 × 10.sup.6                                                                2.1 × 10.sup.8                                                                 266.6                                                                              5.91                                                                             13.2                                         10.sup.5                                                                            20%  36.6 × 10.sup.6                                                                7 × 20.sup.8                                                                   287.3                                                                              5.58                                                                             18.5                                               30%  57.7 × 10.sup.6                                                                6.5 × 10.sup.8                                                                 294.9                                                                              5.30                                                                             22.2                                         10.sup.6                                                                            20%  104.8 × 10.sup.6                                                               10.5 × 10.sup.8                                                                291.3                                                                              4.99                                                                             47.2                                               30%  173.0 × 10.sup.6                                                               22.5 × 10.sup.8                                                                339.4                                                                              4.79                                                                             71.1                                         10.sup.7                                                                            20%  3430 × 10.sup.6                                                                150 × 10.sup.8                                                                 407.4                                                                              4.88                                                                             27.2                                               30%  5400 × 10.sup.6                                                                550 × 10.sup.8                                                                 471.3                                                                              4.75                                                                             25.9                                         control                  209.13                                                                             6.52                                                                             0.00                                         __________________________________________________________________________     (1) The starter incubated for 14-16 hours at 28° C.                    (2) Plate count.                                                         

EXAMPLE 7

Suggested optimum dough mix using baker's yeast and Lactobacillusfermentum ATCC 3910.

The dough was fermented using the formula:

    ______________________________________                                        Improved lactobacilli starter,                                                                     either 20% or 30%                                        Soft wheat flour, 82% extraction                                                                   100 parts                                                Water                70 parts                                                 Salt                 1.2 parts                                                Dry yeast            either 100/150/200                                                            or 250 mg/kg flour                                       Fermentation time    3 hrs at 28° C.                                                        and 90% rh                                               ______________________________________                                    

An overnight starter fermentation (14-16 hrs.) was prepared according tothe results of the above experiment and used for the present experiment.The results of this example as shown in Table 7 showed that thepreparation of the dough mix with the Lactobacillus cell mass more than500 times the yeast cell mass stimulated yeast growth, and relativelydecreased the Lactobacilli cell mass. The decreasing pH which occurs asthe Lactobacilli grow increased the yeast growth. On the other hand, theamount of lysine excreted in the fermented dough tended to be positivelycorrelated only to the absolute amount of Lactobacillus cell mass in thedough mix.

                                      TABLE 7                                     __________________________________________________________________________    Microbial Development.sup.(3) of Dough Mix                                    Using Different Levels of Both Lactobacillus fermentum and Baker's            Yeast.                                                                        Dry Yeast                            Yeast/                                   Dose       Microbial Population.sup.(2)                                                                            Lactobacillus                            Starter.sup.(1)                                                                    mg/kg Before fermentation                                                                        After fermentation.sup.(3)                                                                 Ratio                                    Level                                                                              flour Lactobacillus                                                                        Yeast Lactobacillus                                                                        Yeast Before                                                                            After                                __________________________________________________________________________    30%  100   1.73 × 10.sup.8                                                                1.92 × 10.sup.5                                                               5.25 × 10.sup.8                                                                12.5 × 10.sup.5                                                               1:900                                                                             1:420                                     150   1.73 × 10.sup.8                                                                2.88 × 10.sup.5                                                               12.4 × 10.sup.8                                                                22.5 × 10.sup.5                                                               1:600                                                                             1:551                                     200   1.73 × 10.sup.8                                                                3.84 × 10.sup.5                                                               29.6 × 10.sup.8                                                                27.5 × 10.sup.5                                                               1:420                                                                             1:1022                                    250   1.73 × 10.sup.8                                                                4.81 × 10.sup.5                                                               13 × 10.sup.8                                                                  30 × 10.sup.5                                                                 1:359                                                                             1:433                                20%  100   1.09 × 10.sup.8                                                                1.92 × 10.sup.5                                                               9.2 × 10.sup.8                                                                 15 × 10.sup.5                                                                 1:567                                                                             1:334                                     150   1.09 × 10.sup.8                                                                2.88 × 10.sup.5                                                               15 × 10.sup.8                                                                  27.5 × 10.sup.5                                                               1:378                                                                             1:545                                     200   1.09 × 10.sup.8                                                                3.84 × 10.sup.5                                                               8.4 × 10.sup.8                                                                 15 × 10.sup.5                                                                 1:283                                                                             1:561                                     250   1.09 × 10.sup.8                                                                4.81 × 10.sup.5                                                               16.5 × 10.sup.8                                                                45 × 10.sup.5                                                                 1:226                                                                             1:366                                __________________________________________________________________________     .sup.(1) Fermented with 10.sup.6 cells/g flour, overnight 14-16 hours at      28° C.                                                                 .sup.(2) Plat count cells/g dry dough.                                        .sup.(3) After 3 hours fermentation time at 28° C.                

The addition of dry yeast doses alone (100 to 250 mgs. dry yeast/kgflour) showed no fermentation activities during the three hoursfermentation time, and no leavening occurred in the dough. The pH andthe free lysine content by bioassay technique showed no different fromthe control experiment.

The combination of dry yeast and the Lactobacillus starter decreased theamount of free lysine in the fermented dough but total lysine remainedconstant. It has been shown that yeast cells utilize free lysine toconvert it to a bound form during growth. Strong leavening (53-73%) wasobtained when 200-250 mg. dry yeast/kg flour was used in the dough mix.From the above results, the most favorable conditions to producehigh-quality fermented dough are:

    ______________________________________                                        Lactobacilli improved starter fermented                                                             20-30 parts                                             with 10.sup.6 cells/g flour                                                   Dry yeast for leavening                                                                             200-250 mg/kg flour                                     Salt                  1.2 parts                                               Water                 Variable                                                Flour (82-87.5 extraction)                                                                          100 parts                                               ______________________________________                                    

The starter should be prepared overnight in a warm place, 20°-25° C.,for 14-16 hours.

EXAMPLE 8

Pilot experiments were conducted in both the Experimental BakingLaboratory, Agricultural Research Center, Ministry of Agriculture andFood Security, and the Meet Okba Baking Plant, Cairo General Company forSouthern Baking Plants, Ministry Supplies, Egypt, to evaluate thesuggested technique.

The process at the Experimental Baking Laboratory is semi-automaticusing 10 to 20 kg batches with the following formula:

    ______________________________________                                        Flour, 82% extraction   70-80 parts                                           Mutant or Soltani (conventional                                                                       20-30 parts                                           starter)                                                                      Water                   55 parts                                              Salt, NaCl              1.2 parts                                             Dry yeast               variable                                              ______________________________________                                    

The ingredients were mixed for ten minutes, divided into 190 g pieces,and flattened mechanically. After three hours fermentation at 28° C. inthe fermentation cabinet (90% relative humidity), the loaves were bakedat 340° C. for 1.5 to 2 minutes. The baked products were subjected to ataste test panel, amino acid analysis, lysine bioassay, and availablelysine determination. The results are shown in Tables 8 and 9.

                  TABLE 8                                                         ______________________________________                                        Quality of Dough Mix Fermented.sup.(1)                                        With Lactobacillus and Baker's Yeast                                          (2)    Dry Yeast                                                              Starter                                                                              dose g/kg          Leavening                                                                             Mg free lysine/g                            Level  flour     pH       %       dry dough                                   ______________________________________                                        30%    100       4.52     43.1    951.53                                             150       4.41     51.8    818.3                                              200       4.39     73.3    794.7                                              250       4.55     58.3    661.4                                       20%    100       4.83     28.4    532.9                                              150       4.64     41.38   276.1                                              200       4.90     60.76   299.4                                              250       4.42     30.04   350                                         0.0    100       5.95     0       209.44                                             150       5.93     0       170.65                                             200       5.99     0       187.3                                              250       5.96     7.5     209.48                                      30%    0.00      4.86     49.3    409.3                                       0.0    0.00      6.30     0       210.3                                       ______________________________________                                         .sup.(1) Fermentation 3 hours at 28° C.                                .sup.(2) Starter fermented overnight with 10.sup.6 cells/g flour at           28° C.                                                            

                                      TABLE 9                                     __________________________________________________________________________    Free Available Lysine.sup.(1) in the Improved and Unimproved Baladi           Bread                                                                                              Water                                                                             Available                                                                           % Increase    % Increase                       Treatment            %   Lysine                                                                              Over Control                                                                         Free Lysine                                                                          Over Control                     __________________________________________________________________________    Improved bread       60  6.24  6.70   5.87   146.6                            Improved bread + 100 mg dry yeast/g flour                                                          70  7.24  23.0   5.13   115.5                            Improved bread + 100 mg dry yeast/g flour                                                          60  6.55  11.97  5.03   111.5                            Improved bread + 250 mg dry yeast/g flour                                                          70.90                                                                             7.28  23.59  6.12   157.1                            Unimproved bread (control)                                                                         70-80                                                                             5.85  0      2.38   0                                __________________________________________________________________________     .sup.(1) By bioassay method, mg lysine/100 g protein.                    

EXAMPLE 9 Taste Test Panel

Three types of loaves, obtained by using (1) dry yeast (0.3%); (2) dryyeast (0.1%), plus Lactobacillus; and (3) wild type bacterialfermentation, were subjected to a panel test. The score data includesfour main characteristics: general appearance, color, aroma, and taste.

The scores of the main flour evaluation characteristics were used toobtain a single parameter called quality index value and are reported inthe table below:

    ______________________________________                                        Sample                          General  Quality                              No.       Color   Taste   Aroma Appearance                                                                             Index                                ______________________________________                                        1     A       9.75    7.75  8.25  8.75     8.33                               2     B       9.0     8.5   8.0   8.0      8.48                               3     C       7.0     7.0   6.5   8.0      7.06                               ______________________________________                                    

The results above reveal that Samples No. A and B, in which dry yeast(0.3%) and dry yeast (0.1%) plus Lactobacillus were superior whencompared to traditional wild type (Sample No. C) fermentation. Thetexture was very smooth, and the improved bread kept its fresh appeallonger than the conventional bread.

EXAMPLE 10

The Meet Okba Baking Plant, a government-owned unit, utilizing thetraditional Baladi bread technique, was selected for the next step. Thebaking process there is primarily conducted by hand. Batches were 100 kgeach, and the starter was prepared according to the following formula:

    ______________________________________                                        Baladi flour, 82% ext.                                                                           20 kg                                                      Mutant bacteria/or 10.sup.6 to 10.sup.7 cells/g flour                         Soltani starter    10-12 unbaked loaves                                                          from previous                                                                 conventional batch                                         Warm water         10 to 11 liters                                            ______________________________________                                    

The ingredients were mixed carefully with a low speed blender and thencovered with linen cloths and fermented overnight in a warm place(24°-28° C.). On the next day, the Baladi bread was prepared accordingto the formula:

    ______________________________________                                        Baladi flour, 82% ext.                                                                              100 kg                                                  Overnight starter of the                                                                            20 to 30 kg                                             Mutant or Soltani                                                             Warm water            75 to 90 liters                                         Dry yeast             25 g                                                    Salt, NaCl            1.2 kg                                                  ______________________________________                                    

The ingredients were mixed well for 15 to 20 minutes and then coveredwith linen cloths for 90 minutes at room temperature (24° to 28° C.).The fermented dough was divided by hand into 190 g pieces and proofed ona dusted board for another 90 minutes at room temperature. The doughpieces were flattened by hand and baked at 400°-450° C. for 1 to 1.5minutes.

It is important to note from Table 9 the importance of the effect of thewater content in the dough mix on the amount of available lysineproduced during fermentation. Higher moisture levels facilitate thegrowth of the bacteria during which more lysine is produced.

Utilizing the above formula, process, and Lactobacillus fermentum lysineexcretor with the addition of a very small dose of dry yeast (25 g/100kg) as a leavening agent, it was found that an excellent product couldbe made. The improved Baladi bread was highly accepted by professionalpersonnel (baking industry), nutritionists, technologists, andconsumers. The improved Baladi bread is characterized by its flavor andoverall appeal (crust, color, uniformity). The processing conditionsgave a perfect fermentation which resulted in ideal pocket separationwith a uniform interior. Both the upper and the lower crusts had almostthe same thickness. The crumb was soft and uniform in texture and lightin color. Amino acid analysis by three different approaches (totallysine, available lysine and free lysine bioassay) showed a significantimprovement (Table 9). Each was increased in all its forms in the breadprotein. The available lysine was increased by 20.1% and 23.6%.

A previous nutritive analysis of the improved Baladi bread to test thebio-availability of the bread proteins is presented in Table 10. Asignificant reduction of the flour protein quality resulted from the useof the natural, unimproved bacteria. The PER (protein efficiency ratio)and growth gain were lowered 17.7% and 27% respectively while the feedconversion rate increased 16.6%. The natural process for baking Baladibread using the natural bacteria gives a bread less nutritional valuethan the flour from which it is baked. Compared to the bread baked withthe natural bacteria, the new lysine excreting bacteria gave a breadwith improved nutritive value. The PER and growth gain were increased24.6% and 36% respectively, and the conversion rate decreased 23%. Theimproved bacteria accomplished this by lysine excretion.

                  TABLE 10                                                        ______________________________________                                        Growth of Rats on Baladi Bread Basic Diets.sup.(1)                                                              Con-                                        Growth                            version %                                   Gain        %*     PER       %**  Rate    ***                                 ______________________________________                                        A.     37.8     27     1.53 + .20                                                                             17.7                                                                              6.98 + 1.2                                                                            -16                               Flour                                                                         control                                                                       B.     29.7      0     1.30 + .25                                                                            0    8.36 + 2.0                                                                            0                                 Bread                                                                         wild                                                                          type                                                                          C.     40.4     36     1.62 + .14                                                                             24.6                                                                              6.42 + .06                                                                            -23                               Bread                                                                         Mutant                                                                        D.     148.1    399    3.19 + .14                                                                            145.4                                                                              3.11 + 0.1                                                                            -62                               Casein                                                                        Control                                                                       p-Values                                                                      A × B × C                                                                       <0.05     <0.01     <0.02                                       A × B   <0.07     <0.036    <0.02                                       A × C   <0.5      <0.30     <0.35                                       B × C   <0.02     <0.005    <0.002                                      ______________________________________                                         *Growth gains over the wild type.                                             **PER (Protein Efficiency Ratio) improvement over wild type.                  ***Conversion rates compared to wild type.                                    .sup.(1) Nutritional studies by Dr. C. W. Newman, Department of Animal an     Range Sciences, Montana State University, Bozeman, Montana, U.S.A.       

The invention has been described herein with reference to certainpreferred embodiments. However, as obvious variations thereon willbecome apparent to those skilled in the art, the invention is not to beconsidered as limited thereto.

What is claimed is:
 1. A composition comprisinga yeast in admixture withan heterofermentative microorganism produced from the bacterial speciesLactobacillus fermentum comprising a spontaneous mutant of said wildtype bacteria, which mutant produces lysine in a bread growth medium ingreater quantity than the quantity of lysine produced by the wild typeLactobacillus fermentum.
 2. A composition according to claim 1 whereinthe heterofermentative microorganism has the identifying characteristicsof Lactobacillus fermentum Lex⁺, ATCC 39910 or ATCC
 39911. 3. Acomposition according to claim 1 wherein the heterofermentativemicroorganism is Lactobacillus fermentum Lex⁺, ATCC
 39910. 4. Acomposition according to claim 1 wherein the heterofermentativemicroorganism is Lactobacillus fermentum Lex⁺, ATCC
 39911. 5. Acomposition according to claim 1 wherein the heterofermentative mixturecomprises a ratio of yeast to microorganism in a cell count basis ofabout 1:100 to 1:1000.
 6. A composition according to claim 1 wherein theheterofermentative microorganism is in freeze-dried culture form.
 7. Amethod of making bread, comprisingmixing flour and water with thecomposition of claim 1; fermenting said mixture under lysine-producingconditions; and baking the bread.
 8. A composition of matter,comprisinga cereal grain mixed with the composition of claim
 1. 9. Thecomposition of claim 8, whereinthe cereal grain is wheat.
 10. Thecomposition of claim 8, whereinthe heterofermentative microorganism isL. fermentum LEX⁺, ATCC
 39910. 11. The composition of claim 8,whereinthe heterofermentative microorganism is L. fermentum LEX⁺, ATCC39911.
 12. The composition of claim 8, containing about 10 to 40 partsof the mixture comprisingabout 10⁶ cells of the heterofermentativemicroorganism/g of cereal grain; and about 175-275 mg of dry yeast/kg ofcereal grain.
 13. A method for enhancing the protein quality of cerealgrain, comprisingadding to said cereal grain the composition of claim 1,and fermenting said cereal grain and said composition underlysine-producing conditions.
 14. The method of claim 13, whereinthecereal grain is wheat.
 15. The method of claim 13, whereintheheterofermentative microorganism is in freeze-dried form.
 16. A breadproduct having enhanced protein quality, obtained by a methodcomprisingadding to a precursor of said bread an innoculum of thecomposition of claim 1; fermenting said precursor and said compositionunder lysine-producing conditions; and baking the bread.
 17. A methodfor enhancing the protein quality of bread produced from a sourdoughstarter, comprisingmixing said sourdough starter with an innoculum ofthe composition of claim 1; fermenting said mixture underlysine-producing conditions; and baking the bread.
 18. A freeze-driedcomposition, comprisingthe composition of claim 1 in freeze-dried form.19. The freeze-dried composition of claim 18, whereintheheterofermentative L. fermentum microorganism is selected from the groupconsisting of L. fermentum LEX⁺, ATCC 39910 and ATCC
 39911. 20. Thefreeze-dried composition of claim 19, whereinthe heterofermentativemicroorganism is L. fermentum LEX⁺, ATCC
 39910. 21. The freeze-driedcomposition of claim 18, whereinthe heterofermentative microorganismmutant is L. fermentum LEX⁺, ATCC
 39911. 22. A composition comprisingacereal grain mixed with an innoculum of the freeze-dried composition ofclaim
 18. 23. The composition of claim 22, whereinthe heterofermentativemicroorganism is selected from the group consisting of L. fermentumLEX⁺, ATCC 39910 and ATCC
 39911. 24. The composition of claim 23,whereinthe heterofermentative microorganism is L. fermentum LEX⁺, ATCC39910.
 25. The composition of claim 23, whereinthe heterofermentativemicroorganism is L. fermentum LEX⁺, ATCC
 39911. 26. The composition ofclaim 22,comprising about 10⁶ to 10⁸ cells of the heterofermentativemicroorganism/gm cereal grain.
 27. The composition of claim 26,whereinthe cereal grain is wheat.
 28. As an article of manufacture, apackage comprisinga yeast starter for making bread, and a freeze driedculture of an heterofermentative microorganism produced from thebacterial species Lactobacillus fermentum comprising a spontaneousmutant of said wild type bacteria, which mutant produces lysine in abread growth medium in greater quantity than the quantity of lysineproduced by the wild type Lactobacillus fermentum.
 29. An article ofmanufacture according to claim 28 wherein the starter comprises amixture of yeast and microorganism in a cell count ratio of about 1:100to 1:1000.
 30. An article of manufacture according to claim 28, whereinthe heterofermentative microorganism has the identifying characteristicsof Lactobacillus fermentum Lex⁺, ATCC 39910 or ATCC 39911.